There is provided a pixel defining layer, a production method thereof, and a display substrate. The pixel defining layer comprises a first lyophobic material layer located on a base substrate, wherein the first lyophobic material layer is repellent to solutions for forming respective layers of a light-emitting diode; the pixel defining layer defines a plurality of openings in an array arrangement; and the first lyophobic material layer has a plurality of wall surfaces facing to each of the openings, wherein at least one wall surface of the plurality of wall surfaces has a slope angle larger than 0 degree and less than 90 degrees.